Multiple displacement amplification

ABSTRACT

Disclosed are compositions and a method for amplification of nucleic acid sequences of interest. The method is based on stand displacement replication of the nucleic acid sequences of interest by multiple primers. In one preferred form of the method, referred to as multiple strand displacement amplification, two sets of primers are used, a right set and a left set. The primers in the right set are complementary to one strand of the nucleic acid molecule to be amplified and the primers in the left set are complementary to the opposite strand. The 5′ end of primers in both sets are distal to the nucleic acid sequence of interest when the primers have hybridized to the nucleic acid sequence molecule to be amplified. Amplification proceeds by replication initiated at each primer and continuing through the nucleic acid sequence of interest. A key feature of this method is the displacement of intervening primers during replication by the polymerase. In another preferred form of the method, referred to as whole genome strand displacement amplification, a random set of primers is used to randomly prime a sample of genomic nucleic acid (or another sample of nucleic acid of high complexity). By choosing a set of primers which are sufficiently random, the primers in the set will be collectively, and randomly, complementary to nucleic acid sequences distributed throughout nucleic acid in the sample. Amplification proceeds by replication with a highly processive polymerase initiated at each primer and continuing until spontaneous termination. A key feature of this method is the displacement of intervening primers during replication by the polymerase. In this way, multiple overlapping copies of the entire genome to be synthesized in a short time.

This application is a continuation of application Ser. No. 09/397,915,filed Sep. 17, 1999, entitled “Multiple Displacement Amplification,” byPaul M. Lizardi, now U.S. Pat. No. 6,280,949, which is a continuation ofapplication Ser. No. 08/946,732, filed October 8, 1997, entitled“Multiple Displacement Amplification,” by Paul M. Lizardi, now U.S. Pat.No. 6,124,120. Application Ser. No. 09/397,915, filed Sep. 17, 1999, andapplication Ser. No. 08/946,732, filed October 8, 1997, are herebyincorporated herein by reference.

BACKGROUND OF THE INVENTION

The disclosed invention is generally in the field of nucleic acidamplification.

A number of methods have been developed for exponential amplification ofnucleic acids. These include the polymerase chain reaction (PCR), ligasechain reaction (LCR), self-sustained sequence replication (3SR), nucleicacid sequence based amplification (NASBA), strand displacementamplification (SDA), and amplification with Qβ replicase (Birkenmeyerand Mushahwar, J. Virological Methods, 35:117-126 (1991); Landegren,Trends Genetics 9:199-202 (1993)).

Current methods of PCR amplification involve the use of two primerswhich hybridize to the regions flanking a nucleic acid sequence ofinterest such that DNA replication initiated at the primers willreplicate the nucleic acid sequence of interest. By separating thereplicated strands from the template strand with a denaturation step,another round of replication using the same primers can lead togeometric amplification of the nucleic acid sequence of interest. PCRamplification has the disadvantage that the amplification reactioncannot proceed continuously and must be carried out by subjecting thenucleic acid sample to multiple cycles in a series of reactionconditions.

A variant of PCR amplification, termed whole genome PCR, involves theuse of random or partially random primers to amplify the entire genomeof an organism in the same PCR reaction. This technique relies on havinga sufficient number of primers of random or partially random sequencesuch that pairs of primers will hybridize throughout the genomic DNA atmoderate intervals. Replication initiated at the primers can then resultin replicated strands overlapping sites where another primer canhybridize. By subjecting the genomic sample to multiple amplificationcycles, the genomic sequences will be amplified. Whole genome PCR hasthe same disadvantages as other forms of PCR.

Another field in which amplification is relevant is RNA expressionprofiling, where the objective is to determine the relativeconcentration of many different molecular species of RNA in a biologicalsample. Some of the RNAs of interest are present in relatively lowconcentrations, and it is desirable to amplify them prior to analysis.It is not possible to use the polymerase chain reaction to amplify thembecause the mRNA mixture is complex, typically consisting of 5,000 to20,000 different molecular species. The polymerase chain reaction hasthe disadvantage that different molecular species will be amplified atdifferent rates, distorting the relative concentrations of mRNAs.

Some procedures have been described that permit moderate amplificationof all RNAs in a sample simultaneously. For example, in Lockhart et al.,Nature Biotechnology 14:1675-1680 (1996), double-stranded cDNA wassynthesized in such a manner that a strong RNA polymerase promoter wasincorporated at the end of each cDNA. This promoter sequence was thenused to transcribe the cDNAs, generating approximately 100 to 150 RNAcopies for each cDNA molecule. This weak amplification system allowedRNA profiling of biological samples that contained a minimum of 100,000cells. However, there is a need for a more powerful amplification methodthat would permit the profiling analysis of samples containing a verysmall number of cells.

Accordingly, there is a need for amplification methods that are lesscomplicated, are more reliable, and produce greater amplification in ashorter time.

It is therefore an object of the disclosed invention to provide a methodof amplifying a target nucleic acid sequence in a continuous, isothermalreaction.

It is another object of the disclosed invention to provide a method ofamplifying an entire genome or other highly complex nucleic acid samplein a continuous, isothermal reaction.

It is another object of the disclosed invention to provide a method ofamplifying a target nucleic acid sequence where multiple copies of thetarget nucleic acid sequence are produced in a single amplificationcycle.

It is another object of the disclosed invention to provide a method ofamplifying a concatenated DNA in a continuous, isothermal reaction.

It is another object of the disclosed invention to provide a kit foramplifying a target nucleic acid sequence in a continuous, isothermalreaction.

It is another object of the disclosed invention to provide a kit foramplifying an entire genome or other highly complex nucleic acid samplein a continuous, isothermal reaction.

SUMMARY OF THE INVENTION

Disclosed are compositions and a method for amplification of nucleicacid sequences of interest. The method is based on strand displacementreplication of the nucleic acid sequences by multiple primers. In onepreferred form of the method, referred to as multiple stranddisplacement amplification (MSDA), two sets of primers are used, a rightset and a left set. Primers in the right set of primers each have aportion complementary to nucleotide sequences flanking one side of atarget nucleotide sequence and primers in the left set of primers eachhave a portion complementary to nucleotide sequences flanking the otherside of the target nucleotide sequence. The primers in the right set arecomplementary to one strand of the nucleic acid molecule containing thetarget nucleotide sequence and the primers in the left set arecomplementary to the opposite strand. The 5′ end of primers in both setsare distal to the nucleic acid sequence of interest when the primers arehybridized to the flanking sequences in the nucleic acid molecule.Preferably, each member of each set has a portion complementary to aseparate and non-overlapping nucleotide sequence flanking the targetnucleotide sequence. Amplification proceeds by replication initiated ateach primer and continuing through the target nucleic acid sequence. Akey feature of this method is the displacement of intervening primersduring replication. Once the nucleic acid strands elongated from theright set of primers reaches the region of the nucleic acid molecule towhich the left set of primers hybridizes, and vice versa, another roundof priming and replication will take place. This allows multiple copiesof a nested set of the target nucleic acid sequence to be synthesized ina short period of time. By using a sufficient number of primers in theright and left sets, only a few rounds of replication are required toproduce hundreds of thousands of copies of the nucleic acid sequence ofinterest. The disclosed method has advantages over the polymerase chainreaction since it can be carried out under isothermal conditions. Nothermal cycling is needed because the polymerase at the head of anelongating strand (or a compatible strand-displacement protein) willdisplace, and thereby make available for hybridization, the strand aheadof it. Other advantages of multiple strand displacement amplificationinclude the ability to amplify very long nucleic acid segments (on theorder of 50 kilobases) and rapid amplification of shorter segments (10kilobases or less). In multiple strand displacement amplification,single priming events at unintended sites will not lead to artifactualamplification at these sites (since amplification at the intended sitewill quickly outstrip the single strand replication at the unintendedsite).

In another preferred form of the method, referred to as whole genomestrand displacement amplification (WGSDA), a random set of primers isused to randomly prime a sample of genomic nucleic acid (or anothersample of nucleic acid of high complexity). By choosing a sufficientlylarge set of primers of random or partially random sequence, the primersin the set will be collectively, and randomly, complementary to nucleicacid sequences distributed throughout nucleic acid in the sample.Amplification proceeds by replication with a highly processivepolymerase initiating at each primer and continuing until spontaneoustermination. A key feature of this method is the displacement ofintervening primers during replication by the polymerase. In this way,multiple overlapping copies of the entire genome can be synthesized in ashort time. The method has advantages over the polymerase chain reactionsince it can be carried out under isothermal conditions. Otheradvantages of whole genome strand displacement amplification include ahigher level of amplification than whole genome PCR (up to five timeshigher), amplification is less sequence-dependent than PCR, and thereare no re-annealing artifacts or gene shuffling artifacts as can occurwith PCR (since there are no cycles of denaturation and re-annealing).

In another preferred form of the method, referred to as multiple stranddisplacement amplification of concatenated DNA (MSDA-CD), fragments ofDNA are first concatenated together, preferably with linkers. Theconcatenated DNA is then amplified by strand displacement synthesis withappropriate primers. A random set of primers can be used to randomlyprime synthesis of the DNA concatemers in a manner similar to wholegenome amplification. As in whole genome amplification, by choosing asufficiently large set of primers of random or partially randomsequence, the primers in the set will be collectively, and randomly,complementary to nucleic acid sequences distributed throughoutconcatenated DNA. If linkers are used to concatenate the DNA fragments,primers complementary to linker sequences can be used to amplify theconcatemers. Synthesis proceeds from the linkers, through a section ofthe concatenated DNA to the next linker, and continues beyond. As thelinker regions are replicated, new priming sites for DNA synthesis arecreated. In this way, multiple overlapping copies of the entireconcatenated DNA sample can be synthesized in a short time.

Following amplification, the amplified sequences can be for any purpose,such as uses known and established for PCR amplified sequences. Forexample, amplified sequences can be detected using any of theconventional detection systems for nucleic acids such as detection offluorescent labels, enzyme-linked detection systems, antibody-mediatedlabel detection, and detection of radioactive labels. A key feature ofthe disclosed a method is that amplification takes place not in cycles,but in a continuous, isothermal replication. This makes amplificationless complicated and much more consistent in output. Strand displacementallows rapid generation of multiple copies of a nucleic acid sequence orsample in a single, continuous, isothermal reaction. DNA that has beenproduced using the disclosed method can then be used for any purpose orin any other method desired. For example, PCR can be used to furtheramplify any specific DNA sequence that has been previously amplified bythe whole genome strand displacement method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of an example of multiple strand displacementamplification (MSDA). Diagramed at the top is a double stranded nucleicacid molecule which contains a nucleic acid of interest (hatched area).Hybridized to the nucleic acid molecule are a right and left set ofprimers. Diagramed in the middle are the multiple strands of replicatednucleic acid being elongated from each primer. The polymerase at the endof each elongating strand displaces the elongating strand of the primerahead of it. Diagramed at the bottom are the multiple strands ofreplicated nucleic acid further elongated. Also shown are the next setsof primers which hybridize to their complementary sites on the newlyreplicated strands. The newly replicated strands are made available forhybridization to the primers through displacement by the polymeraseelongating the following strand.

FIG. 2 is a diagram of an example of whole genome strand displacementamplification (WGSDA). At the top is a diagrammatical representation ofgenomic DNA. Hybridized to the nucleic acid molecule are primers from aset of random or partially random primers (the primer lengths are notintended to be to scale). For simplicity, only a portion of one moleculeof genomic DNA is depicted. Diagramed in the middle are the multiplestrands of replicated nucleic acid being elongated from each primer. Thepolymerase at the end of each elongating strand displaces the elongatingstrand of any primer it encounters. Also shown additional primers fromthe set of random or partially random primers which hybridize tocomplementary sites on the newly replicated strands. The newlyreplicated strands are made available for hybridization to the primersthrough displacement by the polymerase elongating a following strand.Diagramed at the bottom are the multiple strands of replicated nucleicacid further elongated. For simplicity only four of the originallysynthesized strands (two on the upper target sequence strand and two onthe lower target sequence strand) are depicted in the bottom panel.

FIG. 3 is a diagram of an example of multiple strand displacementamplification (MSDA). Diagramed at the top is a double stranded nucleicacid molecule which contains a nucleic acid of interest (hatched area).

Hybridized to the nucleic acid molecule are a right set of primers (topstrand in top panel) and a left set of primers (bottom strand in toppanel). Diagramed in the middle are the multiple strands of replicatednucleic acid being elongated from each primer. Also shown are the nextsets of primers which hybridize to their complementary sites on thenewly replicated strands. The newly replicated strands are madeavailable for hybridization to the primers through displacement by thepolymerase elongating the following strand. The polymerase at the end ofeach elongating strand displaces the elongating strand of the primerahead of it. Diagramed at the bottom are the multiple strands ofreplicated nucleic acid further elongated. For simplicity only four ofthe originally synthesized strands (two on the upper target sequencestrand and two on the lower target sequence strand) are depicted in thebottom panel.

FIG. 4 is a diagram of an example of multiple strand displacementamplification of concatenated DNA (MSDA-CD). At the top is adiagrammatical representation of DNA concatenated with linkers. In themiddle, primers complementary to linker sequences are hybridized todenatured strands of the concatenated DNA (the linker and primer lengthsare not intended to be to scale). For simplicity, only a portion of onemolecule of concatenated DNA is depicted. Diagramed at the bottom arethe multiple strands of replicated nucleic acid being elongated fromeach primer. The polymerase at the end of each elongating stranddisplaces the elongating strand of any primer it encounters. Also shownare additional primers which hybridize to complementary sites inreplicated linker sequences on the newly replicated strands. The newlyreplicated strands are made available for hybridization to the primersthrough displacement by the polymerase elongating a following strand.

DETAILED DESCRIPTION OF THE INVENTION

The disclosed method makes use of certain materials and procedures whichallow amplification of target nucleic acid sequences and whole genomesor other highly complex nucleic acid samples. These materials andprocedures are described in detail below.

I. MATERIALS

A. Target Sequence

The target sequence, which is the object of amplification, can be anynucleic acid. The target sequence can include multiple nucleic acidmolecules, such as in the case of whole genome amplification, multiplesites in a nucleic acid molecule, or a single region of a nucleic acidmolecule. For multiple strand displacement amplification, generally thetarget sequence is a single region in a nucleic acid molecule or nucleicacid sample. For whole genome amplification, the target sequence is theentire genome or nucleic acid sample. A target sequence can be in anynucleic acid sample of interest. The source, identity, and preparationof many such nucleic acid samples are known. It is preferred thatnucleic acid samples known or identified for use in amplification ordetection methods be used for the method described herein. The nucleicacid sample can be a nucleic acid sample from a single cell. Formultiple strand displacement amplification, preferred target sequencesare those which are difficult to amplify using PCR due to, for example,length or composition. For whole genome amplification, preferred targetsequences are nucleic acid samples from a single cell. For multiplestrand displacement amplification of concatenated DNA the target is theconcatenated DNA. The target sequences for use in the disclosed methodare preferably part of nucleic acid molecules or samples that arecomplex and non-repetitive (with the exception of the linkers inlinker-concatenated DNA and sections of repetitive DNA in genomic DNA).

Target Sequences for Multiple Strand Displacement Amplification:Although multiple sites in a nucleic acid sample can be amplifiedsimultaneously in the same MSDA reaction, for simplicity, the followingdiscussion will refer to the features of a single nucleic acid sequenceof interest which is to be amplified. This sequence is referred to belowas a target sequence. It is preferred that a target sequence for MSDAinclude two types of target regions, an amplification target and ahybridization target. The hybridization target includes the sequences inthe target sequence that are complementary to the primers in a set ofprimers. The amplification target is the portion of the target sequencewhich is to be amplified. For this purpose, the amplification target ispreferably downstream of, or flanked by the hybridization target(s).There are no specific sequence or structural requirements for choosing atarget sequence. The hybridization target and the amplification targetwithin the target sequence are defined in terms of the relationship ofthe target sequence to the primers in a set of primers. The primers aredesigned to match the chosen target sequence. The top panel of FIG. 1illustrates an example of how primers in a primer set can define theregions in a target sequence. Although preferred, it is not requiredthat sequence to be amplified and the sites of hybridization of theprimers be separate since sequences in and around the sites where theprimers hybridize will be amplified. An example of this is illustratedin FIG. 3.

In multiple strand displacement amplification of linker-concatenatedDNA, the DNA fragments joined by the linkers are the amplificationtargets and the linkers are the hybridization target. The hybridizationtargets (that is, the linkers) include the sequences that arecomplementary to the primers used for amplification. A preferred form ofconcatenated DNA for amplification is concatenated cDNA.

B. Primers

Primers for use in the disclosed amplification method areoligonucleotides having sequence complementary to the target sequence.This sequence is referred to as the complementary portion of the primer.The complementary portion of a primer can be any length that supportsspecific and stable hybridization between the primer and the targetsequence. Generally this is 10 to 35 nucleotides long, but is preferably16 to 24 nucleotides long. For whole genome amplification, it ispreferred that the primers are from 12 to 60 nucleotides long.

It is preferred that primers also contain additional sequence at the 5′end of the primer that is not complementary to the target sequence. Thissequence is referred to as the non-complementary portion of the primer.The non-complementary portion of the primer, if present, serves tofacilitate strand displacement during DNA replication. Thenon-complementary portion of the primer can also include a functionalsequence such as a promoter for an RNA polymerase. The non-complementaryportion of a primer may be any length, but is generally 1 to 100nucleotides long, and preferably 4 to 8 nucleotides long. The use of anon-complementary portion is not preferred when random or partiallyrandom primers are used for whole genome amplification.

Primers for Multiple Strand Displacement Amplification: In the case ofmultiple strand displacement amplification, the complementary portion ofeach primer is designed to be complementary to the hybridization targetin the target sequence. In a set of primers, it is preferred that thecomplementary portion of each primer be complementary to a differentportion of the target sequence. It is more preferred that the primers inthe set be complementary to adjacent sites in the target sequence. It isalso preferred that such adjacent sites in the target sequence are alsoadjacent to the amplification target in the target sequence.

It is preferred that, when hybridized to a target sequence, the primersin a set of primers are separated from each other. It is preferred that,when hybridized, the primers in a set of primers are separated from eachother by at least 5 bases. It is more preferred that, when hybridized,the primers in a set of primers are separated from each other by atleast 10 bases. It is still more preferred that, when hybridized, theprimers in a set of primers are separated from each other by at least 20bases. It is still more preferred that, when hybridized, the primers ina set of primers are separated from each other by at least 30 bases. Itis still more preferred that, when hybridized, the primers in a set ofprimers are separated from each other by at least 40 bases. It is stillmore preferred that, when hybridized, the primers in a set of primersare separated from each other by at least 50 bases.

It is preferred that, when hybridized, the primers in a set of primersare separated from each other by no more than about 500 bases. It ismore preferred that, when hybridized, the primers in a set of primersare separated from each other by no more than about 400 bases. It isstill more preferred that, when hybridized, the primers in a set ofprimers are separated from each other by no more than about 300 bases.It is still more preferred that, when hybridized, the primers in a setof primers are separated from each other by no more than about 200bases. Any combination of the preferred upper and lower limits ofseparation described above are specifically contemplated, including allintermediate ranges. The primers in a set of primers need not, whenhybridized, be separated from each other by the same number of bases. Itis preferred that, when hybridized, the primers in a set of primers areseparated from each other by about the same number of bases.

The optimal separation distance between primers will not be the same forall DNA polymerases, because this parameter is dependent on the netpolymerization rate. A processive DNA polymerase will have acharacteristic polymerization rate which may range from 5 to 300nucleotides per second, and may be influenced by the presence or absenceof accessory ssDNA binding proteins and helicases. In the case of anon-processive polymerase, the net polymerization rate will depend onthe enzyme concentration, because at higher concentrations there aremore re-initiation events and thus the net polymerization rate will beincreased. An example of a processive polymerase is φ29 DNA polymerase,which proceeds at 50 nucleotides per second. An example of anon-processive polymerase is Vent exo(−) DNA polymerase, which will giveeffective polymerization rates of 4 nucleotides per second at lowconcentration, or 16 nucleotides per second at higher concentrations.

To obtain an optimal yield in an MSDA reaction, the primer spacing ispreferably adjusted to suit the polymerase being used. Long primerspacing is preferred when using a polymerase with a rapid polymerizationrate. Shorter primer spacing is preferred when using a polymerase with aslower polymerization rate. The following assay can be used to determineoptimal spacing with any polymerase. The assay uses sets of primers,with each set made up of 5 left primers and 5 right primers. The sets ofprimers are designed to hybridize adjacent to the same target sequencewith each of the different sets of primers having a different primerspacing. The spacing is varied systematically between the sets ofprimers in increments of 25 nucleotides within the range of 25nucleotides to 400 nucleotides (the spacing of the primers within eachset is the same). A series of reactions are performed in which the sametarget sequence is amplified using the different sets of primers. Thespacing that produces the highest experimental yield of DNA is theoptimal primer spacing for the specific DNA polymerase, or DNApolymerase plus accessory protein combination being used.

DNA replication initiated at the sites in the target sequence where theprimers hybridize will extend to and displace strands being replicatedfrom primers hybridized at adjacent sites. Displacement of an adjacentstrand makes it available for hybridization to another primer andsubsequent initiation of another round of replication. The region(s) ofthe target sequence to which the primers hybridize is referred to as thehybridization target of the target sequence. The top panel of FIG. 1illustrates one of the preferred relationships of a set of primers to atarget sequence and to the amplification target of the target sequence.

A set of primers can include any desired number of primers of differentnucleotide sequence. For MSDA, it is preferred that a set of primersinclude a plurality of primers. It is more preferred that a set ofprimers include 3 or more primers. It is still more preferred that a setof primers include 4 or more, 5 or more, 6 or more, or 7 or moreprimers. In general, the more primers used, the greater the level ofamplification that will be obtained. There is no fundamental upper limitto the number of primers that a set of primers can have. However, for agiven target sequence, the number of primers in a set of primers willgenerally be limited to number of hybridization sites available in thetarget sequence. For example, if the target sequence is a 10,000nucleotide DNA molecule and 20 nucleotide primers are used, there are500 non-overlapping 20 nucleotide sites in the target sequence. Evenmore primers than this could be used if overlapping sites are eitherdesired or acceptable. It is preferred that a set of primers include nomore than about 300 primers. It is preferred that a set of primersinclude no more than about 200 primers. It is still more preferred thata set of primers include no more than about 100 primers. It is morepreferred that a set of primers include no more than about 50 primers.It is most preferred that a set of primers include from 7 to about 50primers. Any combination of the preferred upper and lower limits for thenumber of primers in a set of primers described above are specificallycontemplated, including all intermediate ranges.

A preferred form of primer set for use in MSDA includes two sets ofprimers, referred to as a right set of primers and a left set ofprimers. The right set of primers and left set of primers are designedto be complementary to opposite strands of a target sequence. It ispreferred that the complementary portions of the right set primers areeach complementary to the right hybridization target, and that each iscomplementary to a different portion of the right hybridization target.It is preferred that the complementary portions of the left set primersare each complementary to the left hybridization target, and that eachis complementary to a different portion of the left hybridizationtarget. The right and left hybridization targets flank opposite ends ofthe amplification target in a target sequence. A preferred form of theserelationships are illustrated in the top panel of FIG. 1. It ispreferred that a right set of primers and a left set of primers eachinclude a preferred number of primers as described above for a set ofprimers. Specifically, it is preferred that a right or left set ofprimers include a plurality of primers. It is more preferred that aright or left set of primers include 3 or more primers. It is still morepreferred that a right or left set of primers include 4 or more, 5 ormore, 6 or more, or 7 or more primers. It is preferred that a right orleft set of primers include no more than about 200 primers. It is morepreferred that a right or left set of primers include no more than about100 primers. It is most preferred that a right or left set of primersinclude from 7 to about 100 primers. Any combination of the preferredupper and lower limits for the number of primers in a set of primersdescribed above are specifically contemplated, including allintermediate ranges. It is also preferred that, for a given targetsequence, the right set of primers and the left set of primers includethe same number of primers. It is also preferred that, for a giventarget sequence, the right set of primers and the left set of primersare composed of primers of similar length and/or hybridizationcharacteristics.

Primers for Whole Genome Strand Displacement Amplification: In the caseof whole genome strand displacement amplification, it is preferred thata set of primers having random or partially random nucleotide sequencesbe used. In a nucleic acid sample of significant complexity, which isthe referred target sequence for WGSDA, specific nucleic acid sequencespresent in the sample need not be known and the primers need not bedesigned to be complementary to any particular sequence. Rather, thecomplexity of the nucleic acid sample results in a large number ofdifferent hybridization target sequences in the sample which will becomplementary to various primers of random or partially random sequence.The complementary portion of primers for use in WGSDA can be fullyrandomized, have only a portion that is randomized, or be otherwiseselectively randomized.

The number of random base positions in the complementary portion ofprimers are preferably from 20% to 100% of the total number ofnucleotides in the complementary portion of the primers. More preferablythe number of random base positions are from 30% to 100% of the totalnumber of nucleotides in the complementary portion of the primers. Mostpreferably the number of random base positions are from 50% to 100% ofthe total number of nucleotides in the complementary portion of theprimers. Sets of primers having random or partially random sequences canbe synthesized using standard techniques by allowing the addition of anynucleotide at each position to be randomized. It is also preferred thatthe sets of primers are composed of primers of similar length and/orhybridization characteristics.

Primers for Multiple Strand Displacement Amplification On ConcatenatedDNA: For multiple strand displacement amplification of concatenated DNA,a set of primers having random or partially random nucleotide sequencescan be used. In a nucleic acid sample of significant complexity, such asDNA concatenated from a mixture of many sequences, specific nucleic acidsequences present in the sample need not be known and the primers neednot be designed to be complementary to any particular sequence. Rather,the complexity of the nucleic acid sample results in a large number ofdifferent hybridization target sequences in the sample which will becomplementary to various primers of random or partially random sequence.The complementary portion of primers for use in MSDA-CD can be fullyrandomized, have only a portion that is randomized, or be otherwiseselectively randomized.

The number of random base positions in the complementary portion ofprimers are preferably from 20% to 100% of the total number ofnucleotides in the complementary portion of the primers. More preferablythe number of random base positions are from 30% to 100% of the totalnumber of nucleotides in the complementary portion of the primers. Mostpreferably the number of random base positions are from 50% to 100% ofthe total number of nucleotides in the complementary portion of theprimers. Sets of primers having random or partially random sequences canbe synthesized using standard techniques by allowing the addition of anynucleotide at each position to be randomized. It is also preferred thatthe sets of primers are composed of primers of similar length and/orhybridization characteristics.

Where the DNA has been concatenated with linkers, amplification can beperformed using primers complementary to sequences in the linkers. Thisis the preferred form of MSDA-CD. It is preferred that the complementaryportion of each primer is designed to be complementary to sequences inthe linkers. It is preferred that primers for use withlinker-concatenated DNA include primers complementary to both strands ofthe linker sequence. This is illustrated in FIG. 4. It is also preferredthat the primers are not complementary to each other. This prevents theprimers from hybridizing to each other. If the linkers used toconcatenate the DNA are sufficiently long, a set of primerscomplementary to different portions of the linker sequence can be used.This is equivalent to the situation in MSDA, and the sets of primers canbe designed and used in the same manner as discussed for MSDA primersets. Random primers can be used to amplify concatenated DNA whether ornot linkers have been used to concatenate the DNA.

It is preferred that the target sequences for use in MSDA, WGSDA, andMSDA-CD are not, or are not part of, nucleic acid molecules made up ofmultiple tandem repeats of a sequence. It is more preferable that thetarget sequences are not, or are not part of, nucleic acid moleculesmade up of multiple tandem repeats of a single sequence. It is mostpreferred that the target sequences are not, or are not part of, nucleicacid molecules made up of multiple tandem repeats of a single sequencethat were synthesized by rolling circle replication. An example of suchtandem repeat DNA made by rolling circle replication is the tandemsequence DNA described in WO 97/19193. DNA concatenated from identicalor nearly identical DNA fragments is not made by rolling circlereplication and so is not a nucleic acid molecule made up of multipletandem repeats of a single sequence that was synthesized by rollingcircle replication. Thus, although it is preferred that the targetsequences are not nucleic acid molecules made up of multiple tandemrepeats of a single sequence, some such target sequences, such as DNAconcatenated from identical or nearly identical DNA fragments, arepreferred over, and to the exclusion of, nucleic acid molecules made upof multiple tandem repeats of a single sequence that are synthesized byrolling circle replication (such as the tandem sequence DNA described inWO 97/19193). It is preferred that target sequences for the disclosedmethod are not produced by the methods described in WO 97/19193.

Detection Tags: The non-complementary portion of a primer can includesequences to be used to further manipulate or analyze amplifiedsequences. An example of such a sequence is a detection tag, which is aspecific nucleotide sequence present in the non-complementary portion ofa primer. Detection tags have sequences complementary to detectionprobes. Detection tags can be detected using their cognate detectionprobes. Detection tags become incorporated at the ends of amplifiedstrands. The result is amplified DNA having detection tag sequences thatare complementary to the complementary portion of detection probes. Ifpresent, there may be one, two, three, or more than three detection tagson a primer. It is preferred that a primer have one, two, three or fourdetection tags. Most preferably, a primer will have one detection tag.Generally, it is preferred that a primer have 10 detection tags or less.There is no fundamental limit to the number of detection tags that canbe present on a primer except the size of the primer. When there aremultiple detection tags, they may have the same sequence or they mayhave different sequences, with each different sequence complementary toa different detection probe. It is preferred that a primer containdetection tags that have the same sequence such that they are allcomplementary to a single detection probe. For some multiplex detectionmethods, it is preferable that primers contain up to six detection tagsand that the detection tag portions have different sequences such thateach of the detection tag portions is complementary to a differentdetection probe. A similar effect can be achieved by using a set ofprimers where each has a single different detection tag. The detectiontags can each be any length that supports specific and stablehybridization between the detection tags and the detection probe. Forthis purpose, a length of 10 to 35 nucleotides is preferred, with adetection tag portion 15 to 20 nucleotides long being most preferred.

Address Tag: Another example of a sequence that can be included in thenon-complementary portion of a primer is an address tag. An address taghas a sequence complementary to an address probe. Address tags becomeincorporated at the ends of amplified strands. The result is amplifiedDNA having address tag sequences that are complementary to thecomplementary portion of address probes. If present, there may be one,or more than one, address tag on a primer. It is preferred that a primerhave one or two address tags. Most preferably, a primer will have oneaddress tag. Generally, it is preferred that a primer have 10 addresstags or less. There is no fundamental limit to the number of addresstags that can be present on a primer except the size of the primer. Whenthere are multiple address tags, they may have the same sequence or theymay have different sequences, with each different sequence complementaryto a different address probe. It is preferred that a primer containaddress tags that have the same sequence such that they are allcomplementary to a single address probe. The address tag portion can beany length that supports specific and stable hybridization between theaddress tag and the address probe. For this purpose, a length between 10and 35 nucleotides long is preferred, with an address tag portion 15 to20 nucleotides long being most preferred.

C. Linkers

As used herein for concatenating DNA, a linker is a small,double-stranded DNA molecule. For MSDA-CD, linkers serve two mainpurposes; facilitating concatenation of DNA fragments and facilitatingamplification. For the first purpose, linkers are generally designed tohave ends compatible with the ends of the DNA fragments to beconcatenated. For example, if the DNA fragments have blunt ends (or theends will be made blunt), blunt ended linkers would be used. For DNAfragments that have been tailed with one or more nucleotides, thelinkers should have a complementary tail. An example of such tailing isthe addition of single adenosine residues to the 3′ ends of cDNA. Forfacilitating amplification, linkers should have one or more sequencescomplementary to primers to be used in MSDA-CD. Such sequences arereferred to as primer complement portions of the linkers. Primercomplement portions of linkers are complementary to complementaryportions of primers. A primer complement portion can have an arbitrarysequence so long as it is complementary to the portion of the intendedprimer. If there are primer complement portions on opposite strands ofthe linker, they should not overlap. The primer can also have one ormore restriction enzyme cleavage sites. Such restriction sites allow theamplified

DNA to be cut into fragments, and preferably into fragments representingthe original DNA fragments which were concatenated. For this purpose, itis preferred that a rare restriction site be used (for example, aneight-base recognition site). An example of the structure of a linker ofthis type is illustrated below.

     Primer 1>         Restriction SiteP-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNT TNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-P                                             <Primer 2

Linkers can also contain one or more promoter sequences. Such promotersequences allow the amplified DNA to be further amplified bytranscription after MSDA-CD. If two promoters are incorporated into thelinker, they are preferably located on different strands of the linker.An example of a linker, having a single protruding thymidine residue atboth 3′ termini, and a phosphate group at both 5′ termini, isillustrated below (P indicates phosphate).

     Primer 1>           Promoter 1>P-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNT TNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-P              <Promoter 2                         <Primer 2

The promoter and primer sequences may be arranged in any order, but thearrangement shown above is preferred. Any number of primers andpromoters may be used. However, it is preferred that, where the DNA tobe concatenated is cDNA, promoters be incorporated into the cDNA as partof the primers used for cDNA synthesis (Lockhart et al.).

D. Detection Labels

To aid in detection and quantitation of nucleic acids amplified usingthe disclosed method, detection labels can be directly incorporated intoamplified nucleic acids or can be coupled to detection molecules. Asused herein, a detection label is any molecule that can be associatedwith amplified nucleic acid, directly or indirectly, and which resultsin a measurable, detectable signal, either directly or indirectly. Manysuch labels for incorporation into nucleic acids or coupling to nucleicacid probes are known to those of skill in the art. Examples ofdetection labels suitable for use in the disclosed method areradioactive isotopes, fluorescent molecules, phosphorescent molecules,enzymes, antibodies, and ligands.

Examples of suitable fluorescent labels include fluorescein (FITC),5,6-carboxymethyl fluorescein, Texas red, nitrobenz-2-oxa-1,3-diazol4-yl(NBD), coumarin, dansyl chloride, rhodamine,4′-6-diamidino-2-phenylinodole (DAPI), and the cyanine dyes Cy3, Cy3.5,Cy5, Cy5.5 and Cy7. Preferred fluorescent labels are fluorescein(5-carboxyfluorescein-N-hydroxysuccirtimide ester) and rhodamine(5,6-etramethyl rhodamine). Preferred fluorescent labels are FITC andthe cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. The absorption andemission maxima, respectively, for these fluors are: FITC (490 nm; 520nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm),Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm; 778 nm), thus allowing theirsimultaneous detection. The fluorescent labels can be obtained from avariety of commercial sources, including Molecular Probes, Eugene, Oreg.and Research Organics, Cleveland, Ohio.

Labeled nucleotides are a preferred form of detection label since theycan be directly incorporated into the amplification products duringsynthesis. Examples of detection labels that can be incorporated intoamplified DNA or RNA include nucleotide analogs such as BrdUrd (Hoy andSchimke, Mutation Research 290:217-230 (1993)), BrUTP (Wansick et al.,J. Cell Biology 122:283-293 (1993)) and nucleotides modified with biotin(Langer et al., Proc. Natl. Acad. Sci. USA 78:6633 (1981)) or withsuitable haptens such as digoxygenin (Kerkhof, Anal. Biochem.205:359-364 (1992)). Suitable fluorescence-labeled nucleotides areFluorescein-isothiocyanate-dUTP, Cyanine-3-dUTP and Cyanine-5-dUTP (Yuet al., Nucleic Acids Res., 22:3226-3232 (1994)). A preferred nucleotideanalog detection label for DNA is BrdUrd (BUDR triphosphate, Sigma), anda preferred nucleotide analog detection label for RNA isBiotin-16-uridine-5′-triphosphate (Biotin-16-dUTP, BoehringherMannheim). Fluorescein, Cy3, and Cy5 can be linked to dUTP for directlabelling. Cy3.5 and Cy7 are available as avidin or anti-digoxygeninconjugates for secondary detection of biotin- or digoxygenin-labelledprobes.

Detection labels that are incorporated into amplified nucleic acid, suchas biotin, can be subsequently detected using sensitive methodswell-known in the art. For example, biotin can be detected usingstreptavidin-alkaline phosphatase conjugate (Tropix, Inc.), which isbound to the biotin and subsequently detected by chemiluminescence ofsuitable substrates (for example, chemiluminescent substrate CSPD:disodium, 3-(4-methoxyspiro-[1,2,-ioxetane-3-2′-(51′-chloro)tricyclo[3.3.1.1^(3.7)]decane]-4-yl) phenyl phosphate; Tropix, Inc.).

A preferred detection label for use in detection of amplified RNA isacridinium-ester-labeled DNA probe (GenProbe, Inc., as described byArnold et al., Clinical Chemistry 35:1588-1594 (1989)). Anacridinium-ester-labeled detection probe permits the detection ofamplified RNA without washing because unhybridized probe can bedestroyed with alkali (Arnold et al. (1989)).

Molecules that combine two or more of these detection labels are alsoconsidered detection labels. Any of the known detection labels can beused with the disclosed probes, tags, and method to label and detectnucleic acid amplified using the disclosed method. Methods for detectingand measuring signals generated by detection labels are also known tothose of skill in the art. For example, radioactive isotopes can bedetected by scintillation counting or direct visualization; fluorescentmolecules can be detected with fluorescent spectrophotometers;phosphorescent molecules can be detected with a spectrophotometer ordirectly visualized with a camera; enzymes can be detected by detectionor visualization of the product of a reaction catalyzed by the enzyme;antibodies can be detected by detecting a secondary detection labelcoupled to the antibody. As used herein, detection molecules aremolecules which interact with amplified nucleic acid and to which one ormore detection labels are coupled.

E. Detection Probes

Detection probes are labeled oligonucleotides having sequencecomplementary to detection tags on amplified nucleic acids. Thecomplementary portion of a detection probe can be any length thatsupports specific and stable hybridization between the detection probeand the detection tag. For this purpose, a length of 10 to 35nucleotides is preferred, with a complementary portion of a detectionprobe 16 to 20 nucleotides long being most preferred. Detection probescan contain any of the detection labels described above. Preferredlabels are biotin and fluorescent molecules. A particularly preferreddetection probe is a molecular beacon. Molecular beacons are detectionprobes labeled with fluorescent moieties where the fluorescent moietiesfluoresce only when the detection probe is hybridized (Tyagi and Kramer,Nature Biotechnol. 14:303-309 (1995)). The use of such probes eliminatesthe need for removal of unhybridized probes prior to label detectionbecause the unhybridized detection probes will not produce a signal.This is especially useful in multiplex assays.

F. Address Probes

An address probe is an oligonucleotide having a sequence complementaryto address tags on primers. The complementary portion of an addressprobe can be any length that supports specific and stable hybridizationbetween the address probe and the address tag For this purpose, a lengthof 10 to 35 nucleotides is preferred, with a complementary portion of anaddress probe 12 to 18 nucleotides long being most preferred. An addressprobe can contain a single complementary portion or multiplecomplementary portions. Preferably, address probes are coupled, eitherdirectly or via a spacer molecule, to a solid-state support. Such acombination of address probe and solid-state support are a preferredform of solid-state detector.

G. Oligonucleotide Synthesis

Primers, detection probes, address probes, and any otheroligonucleotides can be synthesized using established oligonucleotidesynthesis methods. Methods to produce or synthesize oligonucleotides arewell known in the art. Such methods can range from standard enzymaticdigestion followed by nucleotide fragment isolation (see for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989)Chapters 5, 6) to purely synthetic methods, for example, by thecyanoethyl phosphoramidite method using a Milligen or Beckman System1Plus DNA synthesizer (for example, Model 8700 automated synthesizer ofMilligen-Biosearch, Burlington, Mass. or ABI Model 380B). Syntheticmethods useful for making oligonucleotides are also described by Ikutaet al., Ann. Rev. Biochem. 53:323-356 (1984), (phosphotriester andphosphite-triester methods), and Narang et al., Methods Enzymol.,65:610-620 (1980), (phosphotriester method). Protein nucleic acidmolecules can be made using known methods such as those described byNielsen et al., Bioconjug. Chem. 5:3-7 (1994).

Many of the oligonucleotides described herein are designed to becomplementary to certain portions of other oligonucleotides or nucleicacids such that stable hybrids can be formed between them. The stabilityof these hybrids can be calculated using known methods such as thosedescribed in Lesnick and Freier, Biochemistry 34:10807-10815 (1995),McGraw et al., Biotechniques 8:674-678 (1990), and Rychlik el al.,Nucleic Acids Res. 18:6409-6412 (1990).

H. Solid-State Detectors

Solid-state detectors are solid-state substrates or supports to whichaddress probes or detection molecules have been coupled. A preferredform of solid-state detector is an array detector. An array detector isa solid-state detector to which multiple different address probes ordetection molecules have been coupled in an array, grid, or otherorganized pattern.

Solid-state substrates for use in solid-state detectors can include anysolid material to which oligonucleotides can be coupled. This includesmaterials such as acrylamide, cellulose, nitrocellulose, glass,polystyrene, polyethylene vinyl acetate, polypropylene,polymethacrylate, polyethylene, polyethylene oxide, glass,polysilicates, polycarbonates, teflon, fluorocarbons, nylon, siliconrubber, polyanhydrides, polyglycolic acid, polylactic acid,polyorthoesters, polypropylfumerate, collagen, glycosaminoglycans, andpolyamino acids. Solid-state substrates can have any useful formincluding thin films or membranes, beads, bottles, dishes, fibers, wovenfibers, shaped polymers, particles and microparticles. A preferred formfor a solid-state substrate is a microtiter dish. The most preferredform of microtiter dish is the standard 96-well type.

Address probes immobilized on a solid-state substrate allow capture ofthe products of the disclosed amplification method on a solid-statedetector. Such capture provides a convenient means of washing awayreaction components that might interfere with subsequent detectionsteps. By attaching different address probes to different regions of asolid-state detector, different amplification products can be capturedat different, and therefore diagnostic, locations on the solid-statedetector. For example, in a microtiter plate multiplex assay, addressprobes specific for up to 96 different amplified nucleic acids (eachrepresenting a different target sequence amplified via a different setof primers) can be immobilized on a microtiter plate, each in adifferent well. Capture and detection will occur only in those wellscorresponding to amplified nucleic acids for which the correspondingtarget sequences were present in a sample.

Methods for immobilization of oligonucleotides to solid-state substratesare well established. Oligonucleotides, including address probes anddetection probes, can be coupled to substrates using establishedcoupling methods. For example, suitable attachment methods are describedby Pease et al., Proc. Natl. Acad. Sci. USA 91(11):5022-5026 (1994), andKhrapko et al., Mol Biol (Mosk) (USSR) 25:718-730 (1991). A method forimmobilization of 3′-amine oligonucleotides on casein-coated slides isdescribed by Stimpson et al., Proc. Natl. Acad. Sci. USA 92:6379-6383(1995). A preferred method of attaching oligonucleotides to solid-statesubstrates is described by Guo et al., Nucleic Acids Res. 22:5456-5465(1994).

I. Solid-State Samples

Solid-state samples are solid-state substrates or supports to whichtarget sequences have been coupled or adhered. Target sequences arepreferably delivered in a target sample or assay sample. A preferredform of solid-state sample is an array sample. An array sample is asolid-state sample to which multiple different target sequences havebeen coupled or adhered in an array, grid, or other organized pattern.

Solid-state substrates for use in solid-state samples can include anysolid material to which target sequences can be coupled or adhered. Thisincludes materials such as acrylamide, cellulose, nitrocellulose, glass,polystyrene, polyethylene vinyl acetate, polypropylene,polymethacrylate, polyethylene, polyethylene oxide, glass,polysilicates, polycarbonates, teflon, fluorocarbons, nylon, siliconrubber, polyanhydrides, polyglycolic acid, polylactic acid,polyorthoesters, polypropylfumerate, collagen, glycosaminoglycans, andpolyamino acids. Solid-state substrates can have any useful formincluding thin films or membranes, beads, bottles, dishes, slides,fibers, woven fibers, shaped polymers, particles and microparticles.

Preferred forms for a solid-state substrate are microtiter dishes andglass slides. The most preferred form of microtiter dish is the standard96-well type.

Target sequences immobilized on a solid-state substrate allow formationof target-specific amplified nucleic acid localized on the solid-statesubstrate. Such localization provides a convenient means of washing awayreaction components that might interfere with subsequent detectionsteps, and a convenient way of assaying multiple different samplessimultaneously. Amplified nucleic acid can be independently formed ateach site where a different sample is adhered. For immobilization oftarget sequences or other oligonucleotide molecules to form asolid-state sample, the methods described above for can be used.

A preferred form of solid-state substrate is a glass slide to which upto 256 separate target samples have been adhered as an array of smalldots. Each dot is preferably from 0.1 to 2.5 mm in diameter, and mostpreferably around 2.5 mm in diameter. Such microarrays can befabricated, for example, using the method described by Schena et al.,Science 270:487-470 (1995). Briefly, microarrays can be fabricated onpoly-L-lysine-coated microscope slides (Sigma) with an arraying machinefitted with one printing tip. The tip is loaded with 1μ1 of a DNA sample(0.5 mg/ml) from, for example, 96-well microtiter plates and deposited˜0.005 μl per slide on multiple slides at the desired spacing. Theprinted slides can then be rehydrated for 2 hours in a humid chamber,snap-dried at 100° C. for 1 minute, rinsed in 0.1% SDS, and treated with0.05% succinic anhydride prepared in buffer consisting of 50%1-methyl-2-pyrrolidinone and 50% boric acid. The DNA on the slides canthen be denatured in, for example, distilled water for 2 minutes at 90°C. immediately before use. Microarray solid-state samples can scannedwith, for example, a laser fluorescent scanner with acomputer-controlled XY stage and a microscope objective. A mixed gas,multiline laser allows sequential excitation of multiple fluorophores.

J. DNA Polymerases

DNA polymerases useful in the multiple displacement amplification mustbe capable of displacing, either alone or in combination with acompatible strand displacement factor, a hybridized strand encounteredduring replication. Such polymerases are referred to herein as stranddisplacement DNA polymerases. It is preferred that a strand displacementDNA polymerase lack a 5′ to 3′ exonuclease activity. Strand displacementis necessary to result in synthesis of multiple copies of a targetsequence. A 5′ to 3′ exonuclease activity, if present, might result inthe destruction of a synthesized strand. It is also preferred that DNApolymerases for use in the disclosed method are highly processive. Thesuitability of a DNA polymerase for use in the disclosed method can bereadily determined by assessing its ability to carry out stranddisplacement replication. Preferred strand displacement DNA polymerasesare Bst large fragment DNA polymerase (Exo(-) Bst; Aliotta et al.,Genet. Anal. (Netherlands) 12:185-20 195 (1996)) and exo(-)Bca DNApolymerase (Walker and Linn, Clinical Chemistry 42:1604-1608 (1996)).Other useful polymerases include bacteriophage φ29 DNA polymerase (U.S.Pat. Nos. 5,198,543 and 5,001,050 to Blanco et al.), phage M2 DNApolymerase (Matsumoto et al., Gene 84:247 (1989)), phage φPRD1 DNApolymerase (Jung et al., Proc. Natl. Acad. Sci. USA 84:8287 (1987)),exo(-)VENT® DNA polymerase (Kong et al., J. Biol. Chem. 268:1965-1975(1993)), Klenow fragment of DNA polymerase I (Jacobsen el al., Eur. J.Biochem. 45:623-627 (1974)), T5 DNA polymerase (Chatterjee et al., Gene97:13-19 (1991)), Sequenase (U.S. Biochemicals), PRDl DNA polymerase(Zhu and Ito, Biochim. Biophys. Acta. 1219:267-276 (1994)), and T4 DNApolymerase holoenzyme (Kaboord and Benkovic, Curr. Biol. 5:149-157(1995)). Exo(-)Bst DNA polymerase is most preferred.

Strand displacement can be facilitated through the use of a stranddisplacement factor, such as helicase. It is considered that any DNApolymerase that can perform strand displacement replication in thepresence of a strand displacement factor is suitable for use in thedisclosed method, even if the DNA polymerase does not perform stranddisplacement replication in the absence of such a factor. Stranddisplacement factors useful in strand displacement replication includeBMRF1 polymerase accessory subunit (Tsurumi et al., J. Virology67(12):7648-7653 (1993)), adenovirus DNA-binding protein (Zijderveld andvan der Vliet, J. Virology 68(2):1158-1164 (1994)), herpes sirnplexviral protein ICP8 (Boehmer and Lehnan, J. Virology 67(2):711-715(1993); Skaliter and Lehman, Proc. Natl. Acad. Sci. USA91(22):10665-10669 (1994)); single-stranded DNA binding proteins (SSB;Rigler and Romano, J. Biol. Chem. 270:8910-8919 (1995)); phage T4 gene32 protein (Villemain and Giedroc, Biochemistry 35:14395-14404 (1996);and calf thymus helicase (Siegel et al., J. Biol. Chem. 267:13629-13635(1992)).

The ability of a polymerase to carry out strand displacement replicationcan be determined by using the polymerase in a strand displacementreplication assay such as those described in Examples 1 and 2. The assayin the examples can be modified as appropriate. For example, a helicasecan be used instead of SSB. Such assays should be performed at atemperature suitable for optimal activity for the enzyme being used, forexample, 32° C. for φ29 DNA polymerase, from 46° C. to 64° C. for exo(−)Bst DNA polymerase, or from about 60° C. to 70° C. for an enzyme from ahyperthermophylic organism. For assays from 60° C. to 70° C., primerlength may be increased to 20 bases for random primers, or to 22 basesfor specific primers. Another useful assay for selecting a polymerase isthe primer-block assay described in Kong et al., J. Biol. Chem.268:1965-1975 (1993). The assay consists of a primer extension assayusing an M13 ssDNA template in the presence or absence of anoligonucleotide that is hybridized upstream of the extending primer toblock its progress. Enzymes able to displace the blocking primer in thisassay are useful for the disclosed method.

The materials described above can be packaged together in any suitablecombination as a kit useful for performing the disclosed method.

II. METHOD

The disclosed method is based on strand displacement replication of thenucleic acid sequences by multiple primers. The method can be used toamplify one or more specific sequences (multiple strand displacementamplification), an entire genome or other DNA of high complexity (wholegenome strand displacement amplification), or concatenated DNA (multiplestrand displacement amplification of concatenated DNA). The disclosedmethod generally involves hybridization of primers to a target nucleicacid sequence and replication of the target sequence primed by thehybridized primers such that replication of the target sequence resultsin replicated strands complementary to the target sequence. Duringreplication, the growing replicated strands displace other replicatedstrands from the target sequence (or from another replicated strand) viastrand displacement replication. Examples of such displacement ofreplicated strands are illustrated in the figures. As used herein, areplicated strand is a nucleic acid strand resulting from elongation ofa primer hybridized to a target sequence or to another replicatedstrand. Strand displacement replication refers to DNA replication wherea growing end of a replicated strand encounters and displaces anotherstrand from the template strand (or from another replicated strand).Displacement of replicated strands by other replicated strands is ahallmark of the disclosed method which allows multiple copies of atarget sequence to be made in a single, isothermic reaction.

A. Multiple Strand Displacement Amplification

In one preferred form of the method, referred to as multiple stranddisplacement amplification (MSDA), two sets of primers are used, a rightset and a left set. Primers in the right set of primers each have aportion complementary to nucleotide sequences flanking one side of atarget nucleotide sequence and primers in the left set of primers eachhave a portion complementary to nucleotide sequences flanking the otherside of the target nucleotide sequence. The primers in the right set arecomplementary to one strand of the nucleic acid molecule containing thetarget nucleotide sequence and the primers in the left set arecomplementary to the opposite strand. The 5′ end of primers in both setsare distal to the nucleic acid sequence of interest when the primers arehybridized to the flanking sequences in the nucleic acid molecule.Preferably, each member of each set has a portion complementary to aseparate and non-overlapping nucleotide sequence flanking the targetnucleotide sequence. Amplification proceeds by replication initiated ateach primer and continuing through the target nucleic acid sequence. Akey feature of this method is the displacement of intervening primersduring replication. Once the nucleic acid strands elongated from theright set of primers reaches the region of the nucleic acid molecule towhich the left set of primers hybridizes, and vice versa, another roundof priming and replication will take place. This allows multiple copiesof a nested set of the target nucleic acid sequence to be synthesized ina short period of time.

Multiple strand displacement amplification can be performed by (a)mixing a set of primers with a target sample, to produce anprimer-target sample mixture, and incubating the primer-target samplemixture under conditions that promote hybridization between the primersand the target sequence in the primer-target sample mixture, and (b)mixing DNA polymerase with the primer-target sample mixture, to producea polymerase-target sample mixture, and incubating the polymerase-targetsample mixture under conditions that promote replication of the targetsequence. Strand displacement replication is preferably accomplished byusing a strand displacing DNA polymerase or a DNA polymerase incombination with a compatible strand displacement factor. A preferredexample of MSDA is illustrated in FIG. 1. Another example of MSDA isillustrated in FIG. 3.

By using a sufficient number of primers in the right and left sets, onlya few rounds of replication are required to produce hundreds ofthousands of copies of the nucleic acid sequence of interest. Forexample, it can be estimated that, using right and left primer sets of26 primers each, 200,000 copies of a 5000 nucleotide amplificationtarget can be produced in 10 minutes (representing just four rounds ofpriming and replication). It can also be estimated that, using right andleft primer sets of 26 primers each, 200,000 copies of a 47,000nucleotide amplification target can be produced in 60 minutes (againrepresenting four rounds of priming and replication). These calculationsare based on a polymerase extension rate of 50 nucleotides per second.It is emphasized that reactions are continuous and isothermal—no cyclingis required.

The disclosed method has advantages over the polymerase chain reactionsince it can be carried out under isothermal conditions. No thermalcycling is needed because the polymerase at the head of an elongatingstrand (or a compatible strand-displacement factor) will displace, andthereby make available for hybridization, the strand ahead of it. Otheradvantages of multiple strand displacement amplification include theability to amplify very long nucleic acid segments (on the order of 50kilobases) and rapid amplification of shorter segments (10 kilobases orless). Long nucleic acid segments can be amplified in the disclosedmethod since there no cycling which could interrupt continuous synthesisor allow the formation of artifacts due to rehybridization of replicatedstrands. In multiple strand displacement amplification, single primingevents at unintended sites will not lead to artifactual amplification atthese sites (since amplification at the intended site will quicklyoutstrip the single strand replication at the unintended site).

B. Whole Genome Strand Displacement Amplification

In another preferred form of the method, referred to as whole genomestrand displacement amplification (WGSDA), a random or partially randomset of primers is used to randomly prime a sample of genomic nucleicacid (or another sample of nucleic acid of high complexity). By choosinga sufficiently large set of primers of random or mostly random sequence,the primers in the set will be collectively, and randomly, complementaryto nucleic acid sequences distributed throughout nucleic acid in thesample. Amplification proceeds by replication with a processivepolymerase initiated at each primer and continuing until spontaneoustermination. A key feature of this method is the displacement ofintervening primers during replication by the polymerase. In this way,multiple overlapping copies of the entire genome can be synthesized in ashort time. It can be estimated that, in a WGSDA on a genomic sample,after 180 minutes of incubation each primer will have been elongated by,on average, 55,000 bases. By using a sufficiently high concentration ofprimers, additional priming events on replicated strands will result inadditional rounds of copying. It can be estimated that after 180 minutes400 copies of the entire genome will have been produced.

Whole genome strand displacement amplification can be performed by (a)mixing a set of random or partially random primers with a genomic sample(or other nucleic acid sample of high complexity), to produce anprimer-target sample mixture, and incubating the primer-target samplemixture under conditions that promote hybridization between the primersand the genomic DNA in the primer-target sample mixture, and (b) mixingDNA polymerase with the primer-target sample mixture, to produce apolymerase-target sample mixture, and incubating the polymerase-targetsample mixture under conditions that promote replication of the genomicDNA. Strand displacement replication is preferably accomplished by usinga strand displacing DNA polymerase or a DNA polymerase in combinationwith a compatible strand displacement factor. WGSDA is illustrated inFIG. 2.

The method has advantages over the polymerase chain reaction since itcan be carried out under isothermal conditions. Other advantages ofwhole genome strand displacement amplification include a higher level ofamplification than whole genome PCR (up to 5 times higher),amplification is less sequence-dependent than PCR, and there are nore-annealing artifacts or gene shuffling artifacts as can occur with PCR(since there are no cycles of denaturation and re-annealing).

Following amplification, the amplified sequences can be for any purpose,such as uses known and established for PCR amplified sequences. Forexample, amplified sequences can be detected using any of theconventional detection systems for nucleic acids such as detection offluorescent labels, enzyme-linked detection systems, antibody-mediatedlabel detection, and detection of radioactive labels. A key feature ofthe disclosed method is that amplification takes place not in cycles,but in a continuous, isothermal replication. This makes amplificationless complicated and much more consistent in output. Strand displacementallows rapid generation of multiple copies of a nucleic acid sequence orsample in a single, continuous, isothermal reaction.

It is preferred that the set of primers used for WGSDA be used atconcentrations that allow the primers to hybridize at desired intervalswithin the nucleic acid sample. For example, by using a set of primersat a concentration that allows them to hybridize, on average, every 4000to 8000 bases, DNA replication initiated at these sites will extend toand displace strands being replicated from adjacent sites. It should benoted that the primers are not expected to hybridize to the targetsequence at regular intervals. Rather, the average interval will be ageneral function of primer concentration.

As in multiple strand displacement amplification, displacement of anadjacent strand makes it available for hybridization to another primerand subsequent initiation of another round of replication. The intervalat which primers in the set of primers hybridize to the target sequencedetermines the level of amplification. For example, if the averageinterval is short, adjacent strands will be displaced quickly andfrequently. If the average interval is long, adjacent strands will bedisplaced only after long runs of replication.

In the disclosed method, the DNA polymerase catalyzes primer extensionand strand displacement in a processive strand displacementpolymerization reaction that proceeds as long as desired, generatingmolecules of up to 60,000 nucleotides or larger. Preferred stranddisplacing DNA polymerases are large fragment Bst DNA polymerase (Exo(-)Bst), exo(-)Bca DNA polymerase, the DNA polymerase of the bacteriophageφ29 and Sequenase. During strand displacement replication one mayadditionally include radioactive, or modified nucleotides such asbromodeoxyuridine triphosphate, in order to label the DNA generated inthe reaction. Alternatively, one may include suitable precursors thatprovide a binding moiety such as biotinylated nucleotides (Langer et al.(1981)).

Genome amplification using PCR, and uses for the amplified DNA, isdescribed in Zhang et al., Proc. Natl. Acad. Sci. USA 89:5847-5851(1992), Telenius et al., Genomics 13:718-725 (1992), Cheung et al.,Proc. Natl. Acad. Sci. USA 93:14676-14679 (1996), and Kukasjaarvi etal., Genes, Chromosomes and Cancer 18:94-101 (1997). The uses of theamplified DNA described in these publications are also generallyapplicable to DNA amplified using the disclosed methods. Whole GenomeStrand Displacement Amplification, unlike PCR-based whole genomeamplification, is suitable for haplotype analysis since WGSDA yieldslonger fragments than PCR-based whole genome amplification. PCR-basedwhole genome amplification is also less suitable for haplotype analysissince each cycle in PCR creates an opportunity for priming events thatresult in the association of distant sequences (in the genome) to be puttogether in the same fragment.

C. Multiple Strand Displacement Amplification of Concatenated DNA

In another preferred form of the method, referred to as multiple stranddisplacement amplification of concatenated DNA (MSDA-CD), concatenatedDNA is amplified. A preferred form of concatenated DNA is concatenatedcDNA. Concatenated DNA can be amplified using a random or partiallyrandom set of primers, as in WGSDA, or using specific primerscomplementary to specific hybridization targets in the concatenated DNA.MSDA-CD is preferred for amplification of a complex mixture or sample ofrelatively short nucleic acid samples (that is, fragments generally inthe range of 100 to 6,000 nucleotides). Messenger RNA is the mostimportant example of such a complex mixture. MSDA-CD provides a meansfor amplifying all cDNAs in a cell is equal fashion. Because theconcatenated cDNA can be amplified up to 5,000-fold, MSDA-CD will permitRNA profiling analysis based on just a few cells. To perform MSDA-CD,DNA must first be subjected to a concatenation step. If an RNA sample(such as mRNA) is to be amplified, the RNA is first converted to adouble-stranded cDNA using standard methods. The cDNA, or any other setof DNA fragments to be amplified, is then converted into a DNAconcatenate, preferably with incorporation of linkers.

DNA fragments can be concatenated by ligation using standard conditions.The state of the ends of the DNA fragments, such as blunt, staggered orragged, should be taken into account when concatenating DNA. Forexample, staggered ends, such as those produced by digestion withrestriction enzymes, can be used to mediate concatenation if theoverhanging sequences are compatible. DNA with ragged or staggered endscan be made blunt ended prior to ligation. All of these operations arewell known and of general use. If linkers are used, the linkers caneither be ligated to blunt ended DNA (using blunt ended linkers), or toDNA having compatible overhanging ends, in which case the linkers can bein the form of adaptors.

The following illustrates an example of how the MSDA-CD can be used toamplify mRNA sequences. First, cDNA is made from the mRNA of interest.In this example, the cDNA is made in such a way that it containsphosphorylated 5′-ends. The cDNA is then tailed with a single adenosineresidue at both 3′ ends using Taq DNA polymerase (as described, forexample, by Brownstein et al., Biotechniques 20:1004-1010 (1996), and inthe catalog of Research Genetics, Inc.). The A-tailed cDNA is then mixedwith the T-tailed linkers in the presence of ATP and T4 DNA ligase instandard ligation buffer (see, for example, Holton and Graham, Nucl.Acids Res. 19:1156 (1991), and instructions for the use of pGEM-Tvectors in the Promega Catalog (Promega Biotec, Madison, Wiss., 1997)page 206), and the reaction is incubated overnight at 16° C. to generatelong concatenated DNA molecules. The concatenated molecules consist oftandemly ligated cDNAs and linkers, in alternating order, of thestructure —linker—DNA—linker—DNA—linker—DNA—. The A-tailing andT-tailing method is just one example of many possible methods to obtaintandem, concatenated ligation of linkers and DNA fragments. It is alsopossible to concatenate DNA fragments without linkers to obtainconcatenated molecules of the structure—DNA—DNA—DNA—DNA—. ConcatenatedDNA fragments with linkers is referred to herein as linker-concatenatedDNA or linker-DNA concatenates. Concatenated DNA fragments withoutlinkers is referred to herein as nonlinker-concatenated DNA ornonlinker-DNA concatenates. The terms concatenated DNA and DNAconcatenate refer to both linker-concatenated DNA andnonlinker-concatenated DNA. Amplification of linker-DNA concatenates ismore specific and efficient than amplification of nonlinker-DNAconcatenates, because specific primers can be directed to the linkersequence. Thus, the linker-DNA concatenation method is the preferredform of performing MSDA-CD.

It is preferred that the concatenated product be as long as possible.

This is so because the extent of DNA amplification obtainable withMSDA-CD within any time period is influenced by the length of theconcatenated DNA. The longer the concatenated DNA is, and the morelinkers it contains, the more efficient the amplification process willbe. Concatenation is generally favored by ligating the fragments at highconcentration.

An example of MSDA-CD performed on linker-concatenated DNA isillustrated in FIG. 4. Two different linker-specific primers were usedthat prime on different sequences on different strands of the linker.The two primers should not be complementary to each other. At the top ofFIG. 4 is the double-stranded DNA concatenate with incorporated linkers.The DNA is denatured to make it single-stranded, and the twolinker-specific primers are utilized to amplify the DNA by multiplestrand displacement. It can be estimated that MSDA-CD will amplify a DNAsample as much as 5,000-fold. In the case of the mRNA profiling(Lockhart et al.), MSDA-CD, combined with transcriptional amplification,could be used to improve the limit of detection, permitting profilinganalysis in samples containing only cells.

When using linker-concatenated DNA, multiple strand displacementamplification of concatenated DNA can be performed by (a) mixing primerswith a concatenated DNA sample, to produce an primer-target samplemixture, and incubating the primer-target sample mixture underconditions that promote hybridization between the primers and theconcatenated DNA in the primer-target sample mixture, and (b) mixing DNApolymerase with the primer-target sample mixture, to produce apolymerase-target sample mixture, and incubating the polymerase-targetsample mixture under conditions that promote replication of theconcatenated DNA. Strand displacement replication is preferablyaccomplished by using a strand displacing DNA polymerase or a DNApolymerase in combination with a compatible strand displacement factor.

Following amplification, the amplified sequences can be for any purpose,such as uses known and established for PCR amplified sequences. Forexample, amplified sequences can be detected using any of theconventional detection systems for nucleic acids such as detection offluorescent labels, enzyme-linked detection systems, antibody-mediatedlabel detection, and detection of radioactive labels. A key feature ofthe disclosed method is that amplification takes place not in cycles,but in a continuous, isothermal replication. This makes amplificationless complicated and much more consistent in output. Strand displacementallows rapid generation of multiple copies of a nucleic acid sequence orsample in a single, continuous, isothermal reaction. Sequences in DNAamplified by MSDA-CD performed on concatenated DNA where the linkers orprimers include promoter sequences can be further amplified bytranscriptional amplification using the promoters.

Where the linkers used for concatenation include a restriction enzymesite, the amplified DNA can be fragmented by restriction enzymedigestion.

Cleavage of the amplified DNA can permit or simplify further processingand analysis of the amplified DNA. If the site used appears rarely (forexample, eight-base recognition sites), the resulting fragments willrepresent the original DNA fragments that were concatenated.

When used, a random or partially random set of primers randomly primethe concatenated DNA. By choosing a sufficiently large set of primers ofrandom or mostly random sequence, the primers in the set will becollectively, and randomly, complementary to nucleic acid sequencesdistributed throughout the concatenated DNA. Amplification proceeds byreplication with a processive polymerase initiated at each primer andcontinuing until spontaneous termination. A key feature of this methodis the displacement of intervening primers during replication by thepolymerase. In this way, multiple overlapping copies of the entireconcatenated DNA sample can be synthesized in a short time.

When using random or partially random primers, multiple stranddisplacement amplification of concatenated DNA can be performed by (a)mixing a set of random or partially random primers with a concatenatedDNA sample, to produce an primer-target sample mixture, and incubatingthe primer-target sample mixture under conditions that promotehybridization between the primers and the concatenated DNA in theprimer-target sample mixture, and (b) mixing DNA polymerase with theprimer-target sample mixture, to produce a polymerase-target samplemixture, and incubating the polymerase-target sample mixture underconditions that promote replication of the concatenated DNA. MSDA-CDusing random or partially random primers is similar to WGSDA andproceeds generally as illustrated in FIG. 2.

It is preferred that a set of random or partially random primers usedfor MSDA-CD be used at concentrations that allow the primers tohybridize at desired intervals within the nucleic acid sample. Forexample, by using a set of primers at a concentration that allows themto hybridize, on average, every 4000 to 8000 bases, DNA replicationinitiated at these sites will extend to and displace strands beingreplicated from adjacent sites. It should be noted that the primers arenot expected to hybridize to the target sequence at regular intervals.Rather, the average interval will be a general function of primerconcentration.

As in multiple strand displacement amplification, displacement of anadjacent strand makes it available for hybridization to another primerand subsequent initiation of another round of replication. The intervalat which primers in the set of primers hybridize to the target sequencedetermines the level of amplification. For example, if the averageinterval is short, adjacent strands will be displaced quickly andfrequently. If the average interval is long, adjacent strands will bedisplaced only after long runs of replication. For amplification oflinker-concatenated DNA, where the primers are complementary to linkersequences, the size of the DNA fragments that were concatenateddetermines the spacing between the primers.

D. Modifications And Additional Operations

1. Detection of Amplification Products

Amplification products can be detected directly by, for example, primarylabeling or secondary labeling, as described below.

(a) Primary Labeling

Primary labeling consists of incorporating labeled moieties, such asfluorescent nucleotides, biotinylated nucleotides,digoxygenin-containing nucleotides, or bromodeoxyuridine, during stranddisplacement replication.

For example, one may incorporate cyanine dye UTP analogs (Yu et al.(1994)) at a frequency of 4 analogs for every 100 nucleotides. Apreferred method for detecting nucleic acid amplified in situ is tolabel the DNA during amplification with BrdUrd, followed by binding ofthe incorporated BUDR with a biotinylated anti-BUDR antibody (ZymedLabs, San Francisco, Calif.), followed by binding of the biotin moietieswith Streptavidin-Peroxidase (Life Sciences, Inc.), and finallydevelopment of fluorescence with Fluorescein-tyramide (DuPont de Nemours& Co., Medical Products Dept.).

(b) Secondary Labeling with Detection Probes

Secondary labeling consists of using suitable molecular probes, referredto as detection probes, to detect the amplified DNA or RNA. For example,primer may be designed to contain, in its non-complementary portion, aknown arbitrary sequence, referred to as a detection tag. A secondaryhybridization step can be used to bind detection probes to thesedetection tags. The detection probes may be labeled as described abovewith, for example, an enzyme, fluorescent moieties, or radioactiveisotopes. By using three detection tags per primer, and four fluorescentmoieties per each detection probe, one may obtain a total of twelvefluorescent signals for every replicated strand.

(c) Multiplexing and Hybridization Array Detection

Detection of amplified nucleic acids can be multiplexed by using sets ofdifferent primers, each set designed for amplifying different targetsequences. Only those primers that are able to find their targets willgive rise to amplified products. There are two alternatives forcapturing a given amplified nucleic acid to a fixed position in asolid-state detector. One is to include within the non-complementaryportion of the primers a unique address tag sequence for each unique setof primers. Nucleic acid amplified using a given set of primers willthen contain sequences corresponding to a specific address tag sequence.A second and preferred alternative is to use a sequence present in thetarget sequence as an address tag.

(d) Enzyme-linked Detection

Amplified nucleic acid labeled by incorporation of labeled nucleotidescan be detected with established enzyme-linked detection systems. Forexample, amplified nucleic acid labeled by incorporation ofbiotin-16-UTP (Boehringher Mannheim) can be detected as follows. Thenucleic acid is immobilized on a solid glass surface by hybridizationwith a complementary DNA oligonucleotide (address probe) complementaryto the target sequence (or its complement) present in the amplifiednucleic acid. After hybridization, the glass slide is washed andcontacted with alkaline phosphatase-streptavidin conjugate (Tropix,Inc., Bedford, Mass.). This enzyme-streptavidin conjugate binds to thebiotin moieties on the amplified nucleic acid. The slide is again washedto remove excess enzyme conjugate and the chemiluminescent substrateCSPD (Tropix, Inc.) is added and covered with a glass cover slip. Theslide can then be imaged in a Biorad Fluorimager.

2. Linear Strand Displacement Amplification

A modified form of multiple strand displacement amplification can beperformed which results in linear amplification of a target sequence.This modified method is referred to as linear strand displacementamplification (LSDA) and is accomplished by using a set of primers whereall of the primers are complementary to the same strand of the targetsequence. In LSDA, as in MSDA, the set of primers hybridize to thetarget sequence and strand displacement amplification takes place.However, only one of the strands of the target sequence is replicated.LSDA requires thermal cycling between each round of replication to allowa new set of primers to hybridize to the target sequence. Such thermalcycling is similar to that used in PCR. Unlike linear, or single primer,PCR, however, each round of replication in LSDA results in multiplecopies of the target sequence. One copy is made for each primer used.Thus, if 20 primers are used in LSDA, 20 copies of the target sequencewill be made in each cycle of replication.

DNA amplified using MSDA and WGSDA can be further amplified bytranscription. For this purpose, promoter sequences can be included inthe non-complementary portion of primers used for strand displacementamplification, or in linker sequences used to concatenate DNA forMSDA-CD.

EXAMPLES Example 1 Multiple Strand Displacement Amplification of LambdaDNA

This example illustrates multiple displacement amplification using atotal of 14 primers, 7 in each of a right primer set and a left primerset. The primers in each set are designed to hybridize to oppositestrands on each side of a region to be amplified.

1. The first step is a ligation to close nicks, insuring that longstrands are available for copying. A total of 10 μg of Bacteriophagelambda DNA was dissolved in 100 μl of T4 ligase buffer (10 mM Tris, pH7.5, 0.20 M NaCl, 10 mM MgCl₂, 2 mM ATP). T4 DNA ligase was added to afinal concentration of 8 Units/μl, and the material was incubated for1.5 hours at 37° C. in order to close any nicks in the DNA, making itperfectly double-stranded. The DNA solution was then diluted five-foldwith distilled water, to yield a final DNA concentration of 20 ng/μl.

2. An aliquot of 1.5 μl of ligated lambda DNA (containing 30 ng of DNA)was mixed with 18.2 μl of distilled water, and a suitable multipleprimer mixture (primers made by standard phosphoramidite chemistry). Theprimers used in this example are indicated below. The nomenclature is“PL” for left primers and “PR” for right primers. Seven left primers andseven right primers were used. For each set of 7 primers, the sequencesare spaced 300 to 400 nucleotides between each other. The lambda DNAtargeted by the primers is located within the region demarcated by mappositions 39500 to 22000, and includes a total of approximately 17500bases. This region encompasses lambda Hind II fragments of 2322 bp and9416 bp.

Left Primers (5′ to 3′) 1 GTTGATACATCAACTGCAC PL7 (SEQ ID NO:1) 2CAATTACCTGAAGTCTTTC PL6 (SEQ ID NO:2) 3 TTGTCATATTGTATCATGC PL5 (SEQ IDNO:3) 4 AAGATGAAATAAGAGTAGC PL4 (SEQ ID NO:4) 5 TGCATGCTAGATGCTGATA PL3(SEQ ID NO:5) 6 TATGACTGTACGCCACTGT PL2 (SEQ ID NO:6) 7AGAGTTTCTTTGAGTAATC PL1 (SEQ ID NO:7) Right Primers (5′ to 3′) 1rTTACAACCACTAAACCCAC PR1 (SEQ ID NO:8) 2r AATCGCCAGAGAAATCTAC PR2 (SEQ IDNO:9) 3r AGGGTTATGCGTTGTTCCA PR3 (SEQ ID NO:10) 4r TGTTAAGCAACGCACTCTCPR4 (SEQ ID NO:11) 5r AGTCTGGCGTAACCATCAT PR5 (SEQ ID NO:12) 6rAATAGTGTCTTTTGTGTCC PR6 (SEQ ID NO:13) 7r GCTTGTTACGGTTGATTTC PR7 (SEQID NO:14)

Primers were added at a concentration such that in the following step(step 3, below) the final concentration of each primer was approximately1 micromolar. The lambda DNA and primer mixture was heated at 95° C. for2.5 minutes in order denature the lambda DNA, and the tube wasimmediately placed in ice.

3. The Multiple Strand Displacement Amplification reaction was set up at0° C., in a volume of 30 μl, by adding to the tube of step 2 thefollowing reagents, to give the final concentrations indicated below:

(a) 3 μl of 10X reaction buffer, designed to yield a final concentrationof 40 mM Tris-HCl (pH 7.5), 25 mM NaCl, 8 mM MgCl₂, 6.7 mM DTT, 5% v/vDMSO (dimethylsulfoxide), and 400 μuM mM dATP, dGTP, dCTP, dTTP. SomeMSDA reactions may work better at different concentration of DMSO, inthe range of 1% to 7%.

(b) E. coli single-strand binding protein (SSB) to a final concentrationof 1.4 μM.

(c) Sequenase 2.0 (Amersham Life Sciences) to a concentration of 0.475units/μl (approximately 400 nM).

4. The reaction was incubated at 37° C. for 45 minutes. The DNA wasamplified about 45-fold.

If desired, the amplified DNA can incubated anywhere from 2 to 24 hoursat 55° C. in a buffer containing 30 mM Tris-HCl (pH 8.2), 150 mM NaCl, 1mM EDTA, in order to permit most of the remaining single-strandedmaterial to renature. The amplification yield can be increased by usingmore primers on each side of the DNA region to be-amplified. A suitablenumber of primers for this may be in the range of 10 to 30 primers oneach side of desired DNA domain. Primer numbers exceeding 24 on eachside may increase the frequency of nonspecific amplification.

Example 2 Whole Genome Amplification of Human DNA

This example is for whole genome amplification, as performed for theamplification of the human genome using random primers.

1. DNA was extracted from peripheral blood lymphocytes using a standardproteinase K digestion, followed by extraction with phenol/chloroform.The DNA was quantitated using the Pico-Green dye method (MolecularProbes, Inc., Eugene, Oreg.; Kit P-7589) and the material is thendiluted in TE-0.2 buffer (10 mM Tris pH 8.3, 0.2 mM EDTA), to yield afinal DNA concentration of 1 ng/μl.

2. Four microliters (4 nanograms) of human DNA and 20 μl of TE-0.2buffer were mixed in a 500 μl microcentrifuge tube and denatured at 97°C. for 5 minutes. The tube was then immediately placed in ice.

3. An amplification reaction was set up in an ice bath, in a volume of30 μl, by adding to the tube of step 2 the following reagents, to givethe final concentrations indicated below:

(a) 3 μl of 10X reaction buffer, designed to yield a final concentrationof 25 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH₄)₂ SO₄, 2 mM MgSO₄,0.1% Triton X-100, 5% v/v DMSO (dimethylsulfoxide), and 400 μM mM dATP,dGTP, dCTP, dTTP.

(b) A random DNA oligonucleotide primer of 20 bases in length to a finalconcentration of 4.0 μMolar.

(c) Phage T4 Gene 32 protein added to a final concentration of 30 ng/μl.

(d) Bst DNA polymerase large fragment (New England Biolabs), added last,at a final concentration of 0.35 units/μl.

4. The reaction was incubated at 48° C. for 4 hours, and stopped byaddition of EDTA (final concentration 4 mM).

All publications cited herein are hereby incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

14 19 base pairs nucleic acid single linear DNA NO NO 1 GTTGATACATCAACTGCAC 19 19 base pairs nucleic acid single linear DNA NO NO 2CAATTACCTG AAGTCTTTC 19 19 base pairs nucleic acid single linear DNA NONO 3 TTGTCATATT GTATCATGC 19 19 base pairs nucleic acid single linearDNA NO NO 4 AAGATGAAAT AAGAGTAGC 19 19 base pairs nucleic acid singlelinear DNA NO NO 5 TGCATGCTAG ATGCTGATA 19 19 base pairs nucleic acidsingle linear DNA NO NO 6 TATGACTGTA CGCCACTGT 19 19 base pairs nucleicacid single linear DNA NO NO 7 AGAGTTTCTT TGAGTAATC 19 19 base pairsnucleic acid single linear DNA NO NO 8 TTACAACCAC TAAACCCAC 19 19 basepairs nucleic acid single linear DNA NO NO 9 AATCGCCAGA GAAATCTAC 19 19base pairs nucleic acid single linear DNA NO NO 10 AGGGTTATGC GTTGTTCCA19 19 base pairs nucleic acid single linear DNA NO NO 11 TGTTAAGCAACGCACTCTC 19 19 base pairs nucleic acid single linear DNA NO NO 12AGTCTGGCGT AACCATCAT 19 19 base pairs nucleic acid single linear DNA NONO 13 AATAGTGTCT TTTGTGTCC 19 19 base pairs nucleic acid single linearDNA NO NO 14 GCTTGTTACG GTTGATTTC 19

I claim:
 1. A method of amplifying a target nucleic acid sequence, themethod comprising, bringing into contact a set of primers, DNApolymerase, and a target sample, and incubating the target sample underconditions that promote replication of the target sequence, whereinreplication of the target sequence results in replicated strands,wherein during replication at least one of the replicated strands isdisplaced from the target sequence by strand displacement replication ofanother replicated strand, wherein the set of primers has 3 or moreprimers complementary to the same strand of the target sequence.
 2. Themethod of claim 1 wherein the DNA polymerase is φ29 DNA polymerase. 3.The method of claim 1 wherein the target sequence comprises two strands,wherein the set of primers has 3 or more primers complementary to one ofthe strands of the target sequence and the set of primers also has atleast one primer complementary to the other strand of the targetsequence.
 4. The method of claim 1 wherein the target sequence comprisesan amplification target and a hybridization target, wherein thehybridization target flanks the amplification target, wherein the set ofprimers comprises a plurality of primers, wherein each primer comprisesa complementary portion, wherein the complementary portions of theprimers are each complementary to a different portion of thehybridization target.
 5. The method of claim 1 further comprisingincubating the polymerase-target sample mixture under conditions thatpromote strand displacement.
 6. The method of claim 1 wherein the set ofprimers has 4 or more primers complementary to the same strand of thetarget sequence.
 7. The method of claim 1 wherein the set of primers has4 or more primers.
 8. The method of claim 7 wherein the set of primershas 5 or more primers.
 9. The method of claim 1 wherein the conditionsthat promote replication of the target sequence are substantiallyisothermic.
 10. The method of claim 1 wherein the conditions thatpromote replication of the target sequence do not involve thermalcycling.
 11. The method of claim 1 wherein the conditions do not includethermal cycling.
 12. The method of claim 4 wherein the set of primerscomprises a right set of primers and a left set of primers, wherein thetarget sequence is double-stranded, having a first and a second strand,wherein the hybridization target comprises a right and lefthybridization target, wherein the right hybridization target flanks theamplification target on one end and the left hybridization target flanksthe amplification target on the other end, wherein the complementaryportions of the right set primers are (i) all complementary to the firststrand of the target sequence and (ii) each complementary to a differentportion of the right hybridization target, and wherein the complementaryportions of the left set primers are (i) all complementary to the secondstrand of the target sequence and (ii) each complementary to a differentportion of the left hybridization target.
 13. The method of claim 12wherein the right and left set of primers each have 3 or more primers.14. The method of claim 13 wherein the right and left set of primerseach have 4 or more primers.
 15. The method of claim 14 wherein theright and left set of primers each have 5 or more primers.
 16. Themethod of claim 12 wherein the right and left set of primers each havethe same number of primers.
 17. The method of claim 1 wherein the targetsequence is a nucleic acid sample of substantial complexity, and whereinthe set of primers comprises primers having random nucleotide sequences.18. The method of claim 17 wherein the target sequence is a sample ofgenomic nucleic acid.
 19. The method of claim 17 wherein the primers arefrom 12 to 60 nucleotides in length.
 20. The method of claim 19 whereinthe primers are from 12 to 40 nucleotides in length.
 21. The method ofclaim 20 wherein the primers are from 15 to 40 nucleotides in length.22. The method of claim 21 wherein the primers are from 15 to 25nucleotides in length.
 23. The method of claim 17 wherein the primersare all of the same length.
 24. The method of claim 17 wherein eachprimer comprises a constant portion and a random portion, wherein theconstant portion of each primer has the same nucleotide sequence and therandom portion of each primer has a random nucleotide sequence.
 25. Themethod of claim 1 wherein the target sequence is concatenated DNA. 26.The method of claim 25 wherein the concatenated DNA is concatenated withlinkers.
 27. The method of claim 26 wherein each linker comprises aprimer complement portion, wherein each primer comprises a complementaryportion, wherein the complementary portion of each primer iscomplementary to the complementary portion of the linkers.
 28. Themethod of claim 25 wherein the set of primers comprises primers havingrandom nucleotide sequences.
 29. The method of claim 28 wherein eachprimer comprises a constant portion and a random portion, wherein theconstant portion of each primer has the same nucleotide sequence and therandom portion of each primer has a random nucleotide sequence.
 30. Themethod of claim 25 wherein the concatenated DNA is formed by ligatingDNA fragments together.
 31. The method of claim 30 wherein the DNAfragments are cDNA made from mRNA.
 32. The method of claim 31 whereinthe mRNA comprises a mixture of mRNA isolated from cells.
 33. The methodof claim 1 wherein the target sequence is not a nucleic acid moleculemade up of multiple tandem repeats of a single sequence that wassynthesized by rolling circle replication.
 34. A method of amplifying atarget nucleic acid sequence, the method comprising, (a) mixing a set ofprimers with a target sample, to produce a primer-target sample mixture,and incubating the primer-target sample mixture under conditions thatpromote hybridization between the primers and the target sequence in theprimer-target sample mixture, (b) mixing DNA polymerase with theprimer-target sample mixture, to produce a polymerase-target samplemixture, and incubating the polymerase-target sample mixture underconditions that promote replication of the target sequence, wherein theDNA polymerase is φ29 DNA polymerase, wherein the set of primerscomprises a right set of primers and a left set of primers, wherein thetarget sequence is double-stranded, having a first and a second strand,wherein the right set primers are all complementary to the first strandof the target sequence and the left set primers are all complementary tothe second strand of the target sequence, wherein the right set ofprimers has 4 or more primers and the left set of primers has 4 or moreprimers, wherein replication of the target sequence results inreplicated strands, wherein during replication at least one of thereplicated strands is displaced from the target sequence by stranddisplacement replication of another replicated strand.
 35. A method ofamplifying a target nucleic acid sequence, the method comprising, (a)mixing a set of primers with a target sample, to produce a primer-targetsample mixture, and incubating the primer-target sample mixture underconditions that promote hybridization between the primers and the targetsequence in the primer-target sample mixture, (b) mixing DNA polymerasewith the primer-target sample mixture, to produce a polymerase-targetsample mixture, and incubating the polymerase-target sample mixtureunder conditions that promote replication of the target sequence,wherein the DNA polymerase is φ29 DNA polymerase, wherein replication ofthe target sequence results in replicated strands, wherein duringreplication at least one of the replicated strands is displaced from thetarget sequence by strand displacement replication of another replicatedstrand, wherein the target sequence is a nucleic acid sample ofsubstantial complexity, and wherein the set of primers comprises primershaving random nucleotide sequences.
 36. A method of amplifying a targetnucleic acid sequence, the method comprising, (a) mixing a set ofprimers with a target sample, to produce a primer-target sample mixture,and incubating the primer-target sample mixture under conditions thatpromote hybridization between the primers and the target sequence in theprimer-target sample mixture, (b) mixing DNA polymerase with theprimer-target sample mixture, to produce a polymerase-target samplemixture, and incubating the polymerase-target sample mixture underconditions that promote replication of the target sequence, wherein theDNA polymerase is φ29 DNA polymerase, wherein all of the primers in theset of primers are complementary to the same strand in the targetsequence, wherein the set of primers has 3 or more primers, whereinreplication of the target sequence results in replicated strands,wherein during replication at least one of the replicated strands isdisplaced from the target sequence by strand displacement replication ofanother replicated strand.
 37. A method of amplifying a target nucleicacid sequence, the method comprising, (a) mixing a set of primers with atarget sample, to produce a primer-target sample mixture, and incubatingthe primer-target sample mixture under conditions that promotehybridization between the primers and the target sequence in theprimer-target sample mixture, (b) mixing DNA polymerase with theprimer-target sample mixture, to produce a polymerase-target samplemixture, and incubating the polymerase-target sample mixture underconditions that promote replication of the target sequence, wherein theDNA polymerase is φ29 DNA polymerase, wherein replication of the targetsequence results in replicated strands, wherein during replication atleast one of the replicated strands is displaced from the targetsequence by strand displacement replication of another replicatedstrand, wherein the target sequence is a nucleic acid sample ofsubstantial complexity, and wherein the set of primers comprises primershaving random nucleotide sequences, wherein each primer comprises aconstant portion and a random portion, wherein the constant portion ofeach primer has the same nucleotide sequence and the random portion ofeach primer has a random nucleotide sequence.
 38. A method of amplifyinga target nucleic acid sequence, the method comprising, (a) mixing a setof primers with a target sample, to produce a primer-target samplemixture, and incubating the primer-target sample mixture underconditions that promote hybridization between the primers and the targetsequence in the primer-target sample mixture, (b) mixing DNA polymerasewith the primer-target sample mixture, to produce a polymerase-targetsample mixture, and incubating the polymerase-target sample mixtureunder conditions that promote replication of the target sequence,wherein the DNA polymerase is φ29 DNA polymerase, wherein replication ofthe target sequence results in replicated strands, wherein duringreplication at least one of the replicated strands is displaced from thetarget sequence by strand displacement replication of another replicatedstrand, wherein the conditions that promote replication of the targetsequence do not involve thermal cycling, and wherein the target sequenceis concatenated DNA.
 39. A method of amplifying a target nucleic acidsequence, the method comprising, bringing into contact a set of primers,DNA polymerase, and a target sample, and incubating the target sampleunder conditions that promote replication of the target sequence,wherein replication of the target sequence results in replicatedstrands, wherein during replication at least one of the replicatedstrands is displaced from the target sequence by strand displacementreplication of another replicated strand, wherein the target sequence isa nucleic acid sample of substantial complexity, and wherein the set ofprimers comprises primers having random nucleotide sequences.
 40. Amethod of amplifying a target nucleic acid sequence, the methodcomprising, bringing into contact a set of primers, DNA polymerase, anda target sample, and incubating the target sample under conditions thatpromote replication of the target sequence, wherein all of the primersin the set of primers are complementary to the same strand in the targetsequence, wherein the set of primers has 3 or more primers, whereinreplication of the target sequence results in replicated strands,wherein during replication at least one of the replicated strands isdisplaced from the target sequence by strand displacement replication ofanother replicated strand.
 41. A method of amplifying a target nucleicacid sequence, the method comprising, bringing into contact a set ofprimers, DNA polymerase, and a target sample, and incubating the targetsample under conditions that promote replication of the target sequence,wherein replication of the target sequence results in replicatedstrands, wherein during replication at least one of the replicatedstrands is displaced from the target sequence by strand displacementreplication of another replicated strand, wherein the target sequence isa nucleic acid sample of substantial complexity, and wherein the set ofprimers comprises primers having random nucleotide sequences, whereineach primer comprises a constant portion and a random portion, whereinthe constant portion of each primer has the same nucleotide sequence andthe random portion of each primer has a random nucleotide sequence. 42.A method of amplifying a target nucleic acid sequence, the methodcomprising, bringing into contact a set of primers, DNA polymerase, anda target sample, and incubating the target sample under conditions thatpromote replication of the target sequence, wherein replication of thetarget sequence results in replicated strands, wherein duringreplication at least one of the replicated strands is displaced from thetarget sequence by strand displacement replication of another replicatedstrand, wherein the conditions that promote replication of the targetsequence do not involve thermal cycling, and wherein the target sequenceis concatenated DNA.